High-throughput transcriptomic assays, such as microarrays or RNA-Seq, allow identification of gene expression signatures consisting of hundreds-to-thousands of genes. However, these high-throughput techniques are costly, time-consuming (turnaround time one-to-several days), need centralized processing in many cases, and are not very sensitive in terms of the amount of input material (Katagiri et al., 2009; Nagalakshmi et al., 2010). For example, the hybridization-based, nCounter® System requires ˜100 ng of input total RNA for gene expression studies, with typical assay time 16 hrs (Kulkarni, 2011). In particular, assay sensitivity is becoming an important figure of merit as interest grows in studying minute quantity samples, such as needle biopsies, aspirates and circulating tumor cells (CTCs) (Powell et al., 2012), rather than bulk tissue (Eberwine et al., 2012; Dalerba et al., 2011; Bendall et al., 2012). At the same time, there is no need to use cost- and time-intensive high-throughput techniques in many situations where an assay of several-to-tens of genes will suffice, such as in the case of established biomarker panels (Habel et al., 2006; Colman et al., 2010; Garcia-Bilbao et al., 2012; Mizuarai et al., 2010).
Real-time polymerase chain reaction, also known as quantitative PCR (qPCR), is the golden standard for gene expression-based biomarker assays, due to its sensitivity (single-molecule in the ideal case) and broad dynamic range. However, traditional qPCR is difficult to multiplex, and as a result multi-target experiments require many single reactions to be conducted in parallel, either in microplate format or, in the case of limited-quantity samples, using pre-amplification and proprietary microfluidic platforms—both approaches which add substantial cost, time and complexity to the analysis (Stahlberg et al., 2011; Sanchez-Freire et al., 2012). Multiplexing standard PCR is problematic because target sequences are typically amplified non-uniformly, which results in misrepresentation of low-abundance and/or “difficult” amplicons due to the depletion of reagents (dNTP and primers) and inhibition of polymerase by amplicons (SantaLucia, 2007); another problem is off-target primer binding and primer dimer formation, for which the probability grows as the number of primer pairs in multiplex increases; note that both these effects accumulate over a reaction time course and result in artefacts at a high number of cycles.